Microbial fertilizer regulates C:N:P stoichiometry and alleviates phosphorus limitation in flue-cured tobacco planting soil

Fertilization can be optimized and managed during the flue-cured tobacco growing period by studying the response of soil and microbial biomass stoichiometric characteristics to fertilization. In this study, we investigated the effect of compound fertilizers combined with microbial fertilizer treatments on the stoichiometric characteristics of the rhizosphere soil and the limitations of microbial resources during the flue-cured tobacco growing period. The results indicated that soil and microbial C:N:P varied greatly with the growing period. The effect of sampling time was usually greater than that of fertilization treatment, and microbial C:N:P did not vary with the soil resource stoichiometric ratio. The microbial metabolism of the tobacco-growing soil was limited by phosphorus after extending the growing period, and phosphorus limitation gradually increased from the root extension to the maturation periods but decreased at harvest. The rhizosphere soil microbial nitrogen and phosphorus limitations were mainly affected by soil water content, soil pH, microbial biomass carbon, and the ratio of microbial biomass carbon to microbial biomass phosphorus. Applying microbial fertilizer reduced phosphorus limitation. Therefore, applying microbial fertilizer regulated the limitation of microbial resources by affecting the soil and microbial biomass C:N:P in flue-cured tobacco rhizosphere soils.


Scientific Reports
| (2023) 13:10276 | https://doi.org/10.1038/s41598-023-37438-w www.nature.com/scientificreports/ treatments, including conventional fertilization (CK), conventional fertilization + microbial fertilizer (T1), 75% conventional fertilization + microbial fertilizer (T2), and microbial fertilizer alone (T3). The conventional fertilization treatment was a humic acid organic-inorganic compound fertilizer of 50 g/plant (N + P 2 O 5 + K 2 O ≥33%, 8-5-20) and the microbial fertilizer was 80 g/plant (CociCoLi, Wuhan Kenuo Biotechnology Co., Ltd., Wuhan, China). The number of effective viable bacteria was > 200 million/g, organic matter ≥ 60%, and humic acid ≥ 10%. The microbial fertilizer was used as the base dressing before transplanting and the compound fertilizer was applied at transplant. The test variety was the local main variety K326. Base and top-dressing fertilizer applications, picking, and backing were done in line with local management methods 36 . The row spacing of the tobacco plants was 1.2 × 0.55 m, 1000 plants/acre, and each treatment plot had 60 plants or about 40 m 2 (excluding the protected lines). Rhizosphere soil samples were collected according to the method of Wang et al. 35 during the root extending period (6-8 leaves), the flourishing period (13-14 leaves), the maturation period (3-5 days before harvest), and the harvest period, and named R, F, M, H, respectively. The rhizosphere soils from three similar growing tobacco plants for each fertilization treatment were mixed, sieved to 2 mm after removing impurities, stored in a sealed bag, and transported back to the laboratory for preservation within 24 hours. Each sample was divided into two parts; one was naturally dried to determine basic soil physicochemical properties, and the other was stored at − 20 °C to determine the soil microbial properties.
Soil physical, chemical, and microbial properties. Soil water content (SWC) was calculated by the amount of loss after drying for 48 h using the NY/T1121.3-2006 method. Soil pH was measured in water (1:2.5 w/v) with a pH meter (PHS-3C) according to NY/T1377-2007. The SOC, TN, and TP contents were measured according to HJ 695-2014, NY/T , and NY/T 88-1988, respectively. The soil microbial biomass contents of C, N, and P (MBC, MBN, and MBP) were measured according to the chloroform-fumigationextraction method 40 , and the conversion factor E values of microbial biomass C, N, and P were 0.38, 0.57, and 0.40, respectively [41][42][43][44] . We also calculated a range of soil and microbial ratios, such as SOC/TN (sC/N), SOC/TP (sC/P), TN/TP (sN/P), MBC/MBN (mC/N), MBC/MBP (mC/P), and MBN/MBP (mN/P) in this study.
We measured the activities of four common C, N, and P-related hydrolytic enzymes, including β-1,4glucosidase (BG), β-1,4-N-acetyl-glucosaminidase (NAG), leucine aminopeptidase (LAP), and acid phosphatase (ACP). BG and NAG activities were determined according to a previously described method 45 . LAP and ACP activities were measured using a physiological assay kit (Suzhou Keming Biological Technology Co., Ltd., Suzhou, China) according to the manufacturer's manual. As reported by many studies, BG (NAG + LAP) and ACP were used for C-acquire enzyme activities (C-acq), N-acquire enzyme activities (N-acq), and P-acquire enzyme activities (P-acq) 13 . In addition, we calculated the stoichiometric ratios of C, N, and P microbial enzyme activities, including BG to (NAG + LAP) (eC/N), BG to ACP (eC/P), and (NAG + LAP) to ACP (eN/P) 13 . We also calculated the specific enzyme activity per unit of microbial biomass, such as BG/MBC (C-acq/MBC), (NAG + LAP)/MBN (N-acq/MBN), and ACP/MBP (P-acq/MBP) to represent the microbial enzyme activity coefficient 25 . Finally, we calculated the vector angle and the ratio of C, N, and P enzyme activity to characterize the enzyme stoichiometry 46 , and we calculated microbial stoichiometric homeostasis 7,47,48 . Statistical analysis. We used permutation multivariate analysis of variance (PERMANOVA) to determine the effect and significance of sampling time and the fertilization treatments and their interactions on soil indicators using the "vegan" package in R 49 . We used the "shapiro.test" and "levene.test" packages to test the normality of the distribution and the homogeneity of variance, respectively. A logarithmic or reciprocal transformation was carried out for the indicators that did not conform to a normal distribution. Differences between groups were detected using the Kruskal-Wallis nonparametric test for the indicators that could not be transformed. One-way analysis of variance and Tukey's honestly significant difference (HSD) test were used to determine differences in soil basic physicochemical properties, soil, microbial, and related enzyme C, N, P stoichiometric ratios, and microbial resource limitation-related indicators between the fertilization treatments at the same sampling time 13 . The relationships between the microbial resource limitation (vector angles in this study) and soil physical properties, microbial biomass C, N, and P, and their stoichiometric ratios were analyzed by linear regression using the "ggpmisc" package in R 50 . A heatmap of the correlation coefficients in the "corrplot" package was used to assess the correlation between soil, microbial biomass, and enzymatic C:N:P. Principal component analysis (PCA) was conducted to determine the effects of sampling time and the fertilization treatments on soil microbial biomass and enzymatic C:N:P using the "prcomp" function in R 13 . Statistical analysis and graphing were completed using RStudio software package v.4.2.1.

Results
Effects of different sampling times and fertilization treatments on soil microbial biomass, enzymes, and the C:N:P stoichiometric ratios. SOC, TN and TP were not affected by the interaction between the sampling period and the fertilization treatment, or by either alone (Table 1). TN and TP were highest in the T2 treatment during the H period (Table 2). SWC and soil pH were significantly affected by sampling time and were lowest during the H period. Except for N-acq, all other microbial traits were affected by the sampling time (Table 1). MBC was highest during the F period, and MBN and MBP were highest during the H period ( Table 2).
Only eC/N and eC/P were significantly affected by the interaction between fertilization treatment and sampling time (p < 0.05). mN/P, mC/P, eC/N, eC/P, and eN/P were strongly affected by sampling time (p < 0.05) ( Table 1). sC/P and sN/P were highest in the T2 treatment during the M and H periods ( Fig. 1A-C). mC/N was highest in the CK treatment during the H period (Fig. 1D). mC/P and mN/P were highest in the T3 treatment  Fig. 1E-F). eC/N and eC/P were higher in the T3 treatment than in the other treatments during the M period, but the contents were highest in the T1 treatment during the H period (p < 0.05) (Fig. 1G,H). No significant differences in eN/P were observed among the four treatments during any of the growth periods (p > 0.05) (Fig. 1I). Sampling time and fertilization treatments had no significant effect on soil microbial biomass or soil resources (p > 0.05), indicating soil homeostasis among the different fertilization treatments during the same period (Table 3).

Soil C, N, and P cycle-related enzyme activities and microbial resource limitations. C-acq/
MBC, N-acq/MBN, and P-acq/MBP were significantly affected by sampling time. C-acq/MBC and N-acq/MBN were also affected by the interaction between sampling time and fertilization treatment (p < 0.05) ( Table 1). The C-acq/MBC for flue-cured tobacco was significantly higher in the T3 treatment during the M period (p < 0.05) ( Fig. 2A). The N-acq/MBN and P-acq/MBP ratios were lowest during the H period ( Fig. 2B,C). The vector angles of the four treatments at the different sampling times (p < 0.05) ( Table 1) exceeded 45°, and the order during the M and H periods was T1 > T2 > CK > T3 (Fig. 3A). In contrast, almost all of the soil enzyme stoichiometry points were above the 1:1 line except for some samples from the R period (Fig. 3B), indicating that the samples were P limited except for N limitation during the R period. None of the soils was limited by C and N co-limitation or C and P co-limitation (Fig. 3C). Furthermore, the linear regression analysis shown in Table 4 indicated that the soil vector angle increased with SWC, pH, MBC, and mC/P (p < 0.05).
Correlations between the soil, soil microbial biomass, and enzyme-related C, N, and P stoichiometric ratios. The PCA results showed that axes 1 and 2 explained 25.8% and 24.0%, respectively, of the variation in soil resources, microbial biomass, and enzyme stoichiometry. The differences in the soil and microbial C, N, and P indices at the different sampling times were greater than the differences between fertilization treatments (Fig. 4A,B). The differences during the R and F periods were higher than those during the M Table 1. Permutational multivariate analysis of variance (PERMANOVA) to assess the effects of fertilization treatment, sampling period, and their interactions on soil resources and C, N, and P stoichiometry; and enzymatic angle vectors. SWC, soil water content; pH, soil pH; SOC, soil organic carbon; TN, soil total nitrogen; TP, soil total phosphorus; C-acq, BG; N-acq, NAG + LAP; P-acq, ACP; MBC, microbial biomass C; MBN, microbial biomass N; NBP, microbial biomass P; C-acq/MBC, C related enzyme activity to microbial biomass C; N-acq/MBN, N related enzyme activity to microbial biomass N; P-acq/MBP, P related enzyme activity to microbial biomass P;   (Fig. 4A). The difference in CK was lower than that in the other treatments with added microbial fertilization (Fig. 4B). The correlation analysis further showed no significant relationship between mC/N and sC/N, mC/P and sC/P, or mN/P and sN/P. However, the soil eC/P and eC/N, mC/P and mN/P, and sC/P, and sC/N were positively correlated, as positive correlations were detected between MBC and SOC, MBN and TN, and MBP and TP (Fig. 4C).

Discussion
The stoichiometric balance in soil resources is critical for maintaining microbial metabolism and a dynamic balance among the elements, which reflecting the ability of microorganisms to decompose soil organic matter and release P and indicating the supply of soil nutrients during plant growth 8,28,51 . Consistent with previous studies, soil SOC, TN, and TP contents in this study were significantly positively correlated (p < 0.05), and an interaction was detected between SOC, TN, and TP 28,52 . Tian et al. 28 reported that the mean soil C/N, C/P, and N/P values in China were 11.9, 61, and 5.2, respectively. The average C/N value (11.45) in this study was similar to the above-average value, considering that carbon is a structural element, and its accumulation and consumption are relatively steady 53 . The variability of soil C/N in the different fertilization treatments among sampling times was not significant in this study. The average C/P and N/P values were 14.1 and 1.2, which were lower than the average soil values in China, possibly due to the low organic carbon content in red soil in this study 54 , or the lower pH and N availability 55 . However, adding microbial fertilizer improved the soil C/P and N/P values during the H period (Fig. 1B,C), possibly because Bacillus subtilis was contained in the microbial fertilizer, which improved soil N fixation capacity and SOC content 56 ; Bacillus mucilaginosus decreases soil P content 57 . Interestingly, the reduced usage of compound fertilizer combined with microbial fertilizer (T2) in this study had a larger effect on increasing soil C/P and N/P (Fig. 1B,C).
Microbial resource limitations describe microbial growth and activity that is limited by nutrient availability and energy 58 . Ecological stoichiometry theory suggests that the C:N:P ratio of soil microbial biomass is more stable relative to the soil C, N, and P stoichiometry ratio and reflects the state of microbial C, N, and P demand 59 .
Our results indicate no significant correlation between the microbial biomass stoichiometric ratio and the soil resources stoichiometric ratio (Fig. 4C). The strict homeostasis of soil microbial biomass between the fertilization treatments and different sampling times also confirmed the stability of microbial stoichiometry 7 (Table 3), which supports our first hypothesis. Moreover, the global average values of mC/N, mC/P, and mN/P are 7.6, 42.4, and 5.6, respectively 52 . The mC/P and mN/P values were 30.52 and 2.37 in this study, which was lower than the global levels. This result indicates that soil microorganisms have a weak tendency to assimilate soil available P, and the ability to absorb P results from competition with plants 60 . However, the mC/N value (19.14) was higher than the global level, suggesting a relatively strong N fixation ability of the soil microorganisms in this study 61 . The mC/N value was relatively stable in this study compared with a previous study 1 , and mC/P and mN/P varied more among the sampling periods (Fig. 1A), indicating greater stoichiometric plasticity in microbial P 1 . In contrast to a previous study, Qi et al. 7 showed that soil mC/P and mN/P values were highest during the Table 2. Soil physicochemical properties and biological indicators across treatments during the flue-cured tobacco growing period. SWC, soil water content; pH, soil pH; SOC, soil organic carbon; TN, soil total nitrogen; TP, soil total phosphorus; C-acq, BG; N-acq, NAG + LAP; P-acq, ACP; MBC, microbial biomass C; MBN, microbial biomass N; NBP, microbial biomass P. R, F, M, and H indicate the root extending, flourishing, maturing and harvesting sampling periods, respectively. CK, conventional fertilization; T1, conventional fertilization + microbial fertilizer; T2, 75% conventional fertilization + microbial fertilizer; T3, microbial fertilizer. Values are mean ± standard error (n = 3). Lowercase letters indicate significant differences among the fertilizer treatments for each growing period (Tukey's HSD test, p < 0.05). Previous studies have indicated that the ratio of global soil C, N, and P-related enzyme activities is 1:1:1 20 . A ratio that deviates from 1:1:1 suggests that soil microorganisms are affected by C, N, or P limitations 20 . The C:N:P ratio of the enzyme activities in this study was 1:1.45:1.64, indicating that soil microorganisms were more restricted by N and P than soil C. In addition, the enzyme stoichiometry points were mostly above the 1:1 line, and the vector angles in almost all treatments were greater than 45°, showing that P was limited, except for a few points where N was limited during the R period. Moreover, the soil microorganisms changed from N-limited to P-limited with the extension of the growing period 46 (Fig. 3A). Notably, enzymatic stoichiometry is controversial for determining carbon resource constraints 25,31 . However, our study combined C, N, and P enzyme www.nature.com/scientificreports/ Table 3. Homeostatic coefficients of soil microbial biomass and their stoichiometries. 1/H is the slope of the regression line between ln (y) and ln (x), where x is the soil resource stoichiometric ratio (e.g., sC/N), and y is the microbial biomass carbon, nitrogen, and phosphorus stoichiometric ratio (e.g. mC/N). The regression relationship was not significant (p > 0.05) in this study, so microbial stoichiometry was "strictly homeostatic". R,  www.nature.com/scientificreports/ stoichiometric characteristics and the vector angle to determine microbial resource limitations, took place on tobacco planting soil that was limited by N and P, which can minimize this bias, and yielded convincing results. The soil N and P limitations may be due to the acidic soil in this study. Previous research has suggested that P limitations are mainly due to the strong binding of Fe 3+ and Al 3+ or that water-soluble P is slowly converted to occluded P in acidic soil, resulting in reduced P utilization 62,63 . Secondly, P limitation increased first and then decreased as the growing period of flue-cured tobacco was extended. The T3 treatment had an earlier weakening trend, and weakened from the F to the M period, while the remaining treatments showed a weakening trend from the M to the H period. The changes in P limitation may have occurred because a large amount of P is needed to supply flue-cured tobacco primary productivity during the vigorous growing period, thereby increasing the P limitation of soil microorganisms 13 , and P limitation was alleviated by increasing the soil total P during the H period 64 ( Table 2). The results also show that the full application of microbial fertilizer (T3) had a more obvious effect on alleviating P limitation, which was conducive to the microbial nutrient balance by alleviating competition for nutrients between soil microbes and the soil. Herein, our results support the second and third hypotheses that different fertilizer applications lead to changes in microbial resource limitations, which varied during different growth periods. Moreover, Yang et al. 12 showed that microbial N and P limitations are affected by the soil nutrient stoichiometric ratio, soil water content, soil pH, soil bulk density, and SOC. At the same time, other studies have shown that temperature, soil moisture, soil pH, and SOC affect microbial P limitations 12,65 . In this study, SWC, soil pH, MBC, and mC/P had significant negative effects on the microbial N and P limitations (Table 4). Consistent  stoichiometry (B, C). Vector angles < 45° indicate N limitations, whereas those > 45° indicate P limitations. R, F, M, and H indicate the root extending, flourishing, maturation, and harvesting sampling periods, respectively. CK, conventional fertilization; T1, conventional fertilization + microbial fertilizer; T2, 75% conventional fertilization + microbial fertilizer; T3, microbial fertilizer. Table 4. Linear regression of soil physicochemical and microbial indicators with vector angles. SWC, soil water content; pH, soil pH; SOC, soil organic carbon; TN, soil total nitrogen; TP, soil total phosphorus; C-acq, BG; N-acq, NAG + LAP; P-acq, ACP; MBC, microbial biomass C; MBN, microbial biomass N; MBP, microbial biomass P. Significant values are in bold.

Variables (x)
Regression   A, B) and correlation between the soil physicochemical and microbial indicators (C). R, F, M, and H indicate the root extending, flourishing, maturation, and harvesting sampling periods, respectively. CK, conventional fertilization; T1, conventional fertilization + microbial fertilizer; T2, 75% conventional fertilization + microbial fertilizer; T3, microbial fertilizer. Blue and red represent positive and negative correlations, respectively. The darker the color, the stronger the relationship. *significant at p < 0.05; **significant at p < 0.01; ***significant at p < 0.001.